Abstract( ! Our goal is to develop novel nanoplatforms for efficient in vivo delivery of genome editing machinery into specific cell types and tissues; nanodelivery systems that are non-toxic and can be used for therapeutic genome editing in human. Regarding nanocarrier, we will engineer the innovative hepatitis E viral nanoparticle (HEVNP) developed by the Cheng (one of the PIs) laboratory, such that cell specific targeting/endocytic ligand will be displayed on the viral nanoparticle surface, and genome editing machinery and endosomolytic peptides will be encapsulated inside the core. HEVNP is resistant to gastric acid environment and therefore can deliver payload to intestine via oral route. We hypothesize that through introduction of stealth peptide elements and cell-type specific targeting ligand on the viral capsid proteins, such novel nanocarrier can deliver gene editing machinery to specific cell type, not only via the oral route, but also via the intravenous route. Our goal is to genome edit intestinal adenomatous polyp cells, intestinal epithelial cells, and intestinal stem cells of ApcMin/+ mice (a mouse model of familial adenomatous polyposis) by correcting a mutant Apc tumor suppressor gene, leading to prevention of new polyp formation and regression of existing polyps (size & number). We will employ the enabling one-bead one-compound (OBOC) and one-bead two-compound (OB2C) combinatorial library methods (invented by Dr. Lam, one of the PIs) to develop D-amino acid containing targeting/endocytic ligands against intestinal adenomatous polyp cells, intestinal epithelial cells, and intestinal stem cells of ApcMin/+ mice. We will use the OBOC method to discover membrane active peptides with endosomolytic activities. These endosomolytic peptides will bind to endosome at pH5 but not to plasma membrane at pH7.2. Specific aims are as follows: UG3 (Phase 1) Aim 1: To design and synthesize OB2C/OBOC combinatorial libraries for the discovery of (a) cell- specific targeting and endocytic peptides, and (b) endosomolytic peptides. Aim 2: (a) To engineer hepatitis E viral nanoparticle (HEVNP) with cell-specific targeting ligands, (b) to design genome editing machinery for GFP, luciferase and Apc correction, and (c) to assemble a series of nanoconstructs encapsulating the genome machinery and endosomolytic peptides. Aim 3: To evaluate the genome editing functions of the nanoconstructs from aim 2, in vitro with murine intestinal enterocytes and adenomatous polyp cells, and in vivo (oral & iv) with Apcmin/+ mice. UH3 (Phase 2) Aim 1, 2 & 3: To scale up the production of the genome editing nanodelivery platform. To perform preclinical pharmacology/toxicology studies in mouse and Rhesus macaques. To collaborate with investigator from SCGE Large Animal Testing Centers on validation of the delivery system. !